Evaluation of glutamate excitotoxicity induced by dengue virus neuroadapted D4MB -6

El virus de dengue (DENV), a pesar de ser clasificado como un virus no neurotrópico, induce durante la infección manifestaciones neurológicas como la alteración de la conciencia. Hasta el momento, los signos y síntomas neurológicos que aparecen durante la infección por DENV no se han asociado a un mecanismo en particular. Durante la infección con el JEV, WNV y el HIV se produce la disfunción y muerte de neuronas mediada por procesos excitatorios. Para Dengue, hasta el momento sólo se ha reportado en modelos in vivo e in vitro y en muestras post-mortem el daño del tejido y la perdida neuronal, sin embargo no se conoce si durante la infección con este virus suceden eventos excitotoxicos o si la la exacerbada respuesta inmune afecta la superviviencia neuronal En el presente estudio se planteó -utilizando el modelo de neuroinfección desarollado en nuestro laboratorio-, evaluar las posibles causas de alteración y muerte neuronal inducidas por la cepa D4MB-6 y el efecto de dos fármacos, ácido valproico (VPA) y MK-801 con el fin de. Para esto, se infectaron ratones Balb/C de 7 dpn con el D4MB-6 tratados o no tratados con VPA o MK 801. Los animales fueron observados y pesados diariamente por 3 o 6 dpi y sacrificados para la extracción del encéfalo y médula. De estos tejidos se obtuvieron homogenizados o cortes histológicos para evaluar la infección y producción viral, la morfología, la expresión de algunas proteínas pro y anti-apoptóticos. En los animales infectados no tratados, las manifestaciones clínicas fueron evidentes al 3er dpi y severas al 6to dpi, al igual que las alteraciones histológicas, caracterizadas por apoptosis, necrosis y espongiosis neuronal acompañadas de alteraciones vasculares como hemorragias, edema e infiltrado de células mononucleares. En esta misma, condición se observó astrogliosis, neurodegeneración y aumento en la expresión de proteínas pro-apoptóticas, como Casp 3, 8, y Bax. Por el contrario, en los animales infectados y tratados todas las manifestaciones y alteraciones neurológicas fueron reducidas, detectando sólo algunas células en apoptosis y neurodegeneración en el cerebelo de animales infectados y tratados con VPA y MK 801, al igual que la producción y transcritos pro- apoptóticos. Estos resultados sugieren que el virus D4MB-6 induce encefalitis y mielitis, como alteración vascular, así como muerte neuronal mediada por procesos excitotóxicos e inmunológicos. Dichas alteraciones son prevenidas de forma total o parcial con los fármacos MK 801 y VPA.Magíster en Ciencias Básicas BiomédicasMaestríaThe dengue virus (DENV), despite being classified as a non-neurotropic virus during infection induces neurological symptoms such as impaired consciousness. So far, the neurological signs and symptoms that appear during DENV infection have not been associated to a particular mechanism. During infection with JEV, WNV and HIV dysfunction and neuronal death mediated excitatory process occurs. Dengue, so far only been reported in in vivo and in vitro models and samples postmortem tissue damage and neuronal loss, however not known whether during infection with this virus occur excitotoxic events or the exacerbated immune response affects neuronal survival. In the present study it raised -using neuroinfection model developed in our laboratory-, so evaluate potential causes of impairment and neuronal death induced D4MB-6 strain and the effect of two drugs, valproic acid (VPA) and MK 801. For this, Balb/C mice were infected with D4MB-6 treated or not treated with VPA or MK 801. The animals were observed and weighed daily for 3 or 6 dpi and sacrificed for the removal of the brain and spinal cord. These tissues homogenates or tissue sections to assess viral infection and production, morphology, expression of some pro and anti-apoptotic proteins were obtained. In infected untreated animals, the clinical manifestations were evident to 3rd and severe to the 6th dpi, as well as histological alterations, characterized by apoptosis, neuronal necrosis and spongiosis accompanied by vascular disorders such as bleeding, edema and infiltration of mononuclear cells. In the same, astrogliosis condition, neurodegeneration and increased expression of pro-apoptotic proteins such as Casp3, 8, and Bax it was observed. Conversely, infected and treated at all manifestations and neurological disorders animals were reduced by detecting only a few cells in apoptosis and neurodegeneration in the cerebellum of infected and treated with VPA and MK 801 animals, as the production and transcribed pro- apoptotic. These results suggest that the D4MB-6 virus induces encephalitis and myelitis, and vascular changes and mediated excitotoxic neuronal death and immunological processes. Such alterations are prevented in whole or in part with the MK 801 and VPA drug

A case series of severe dengue with neurological presentation in children from a colombian hyperendemic area

Dengue transmission is sustained in Colombia with increasing prevalence mainly in children. This work aimed to describe a case series of children diagnosed with dengue presenting neurological disease in Huila Province of Colombia. Eleven pediatric febrile patients confirmed for dengue disease and presenting neurological signs were studied in the University Hospital of Neiva, Huila Province. Clinical and laboratory findings, CSF cytochemical analysis, neurology images, and serology and molecular studies were performed. Viral RNA was detected in all patients' sera by RT-PCR. Nine out of 11 were primary infections. Tonic-clonic seizures (73%), consciousness alterations (27%), irritability (27%), and ataxia (18%) were the most frequent neurological signs. None of the patients had plasma leakage, hypovolemic shock, or liver disease, confirming the encephalitis diagnosis. Diagnostic images did not show abnormal findings, but neither bacterial nor fungal infections were detected in CSF analysis. All patients survived without sequelae except for one patient that presented ataxia for months. In conclusion, we described a group of children with neurological signs during severe dengue disease as the main finding, indicating the importance to including dengue as a differential diagnosis in neurological patients from endemic areas.Dengue transmission is sustained in Colombia with increasing prevalence mainly in children. This work aimed to describe a case series of children diagnosed with dengue presenting neurological disease in Huila Province of Colombia. Eleven pediatric febrile patients confirmed for dengue disease and presenting neurological signs were studied in the University Hospital of Neiva, Huila Province. Clinical and laboratory findings, CSF cytochemical analysis, neurology images, and serology and molecular studies were performed. Viral RNA was detected in all patients' sera by RT-PCR. Nine out of 11 were primary infections. Tonic-clonic seizures (73%), consciousness alterations (27%), irritability (27%), and ataxia (18%) were the most frequent neurological signs. None of the patients had plasma leakage, hypovolemic shock, or liver disease, confirming the encephalitis diagnosis. Diagnostic images did not show abnormal findings, but neither bacterial nor fungal infections were detected in CSF analysis. All patients survived without sequelae except for one patient that presented ataxia for months. In conclusion, we described a group of children with neurological signs during severe dengue disease as the main finding, indicating the importance to including dengue as a differential diagnosis in neurological patients from endemic areas

Dengue virus infection of blood-brain barrier cells: consequences of severe disease

More than 500 million people worldwide are infected each year by any of the four-dengue virus (DENV) serotypes. The clinical spectrum caused during these infections is wide and some patients may develop neurological alterations during or after the infection, which could be explained by the cryptic neurotropic and neurovirulent features of flaviviruses like DENV. Using in vivo and in vitro models, researchers have demonstrated that DENV can affect the cells from the blood–brain barrier (BBB) in several ways, which could result in brain tissue damage, neuronal loss, glial activation, tissue inflammation and hemorrhages. The latter suggests that BBB may be compromised during infection; however, it is not clear whether the damage is due to the infection per se or to the local and/or systemic inflammatory response established or activated by the BBB cells. Similarly, the kinetics and cascade of events that trigger tissue damage, and the cells that initiate it, are unknown. This review presents evidence of the BBB cell infection with DENV and the response established toward it by these cells; it also describes the consequences of this response on the nervous tissue, compares these evidence with the one reported with neurotropic viruses of the Flaviviridae family, and shows the complexity and unpredictability of dengue and the neurological alterations induced by it. Clinical evidence and in vitro and in vivo models suggest that this virus uses the bloodstream to enter nerve tissue where it infects the different cells of the neurovascular unit. Each of the cell populations respond individually and collectively and control infection and inflammation, in other cases this response exacerbates the damage leaving irreversible sequelae or causing death. This information will allow us to understand more about the complex disease known as dengue, and its impact on a specialized and delicate tissue like is the nervous tissue

Isolation of the Human Cytomegalovirus from bodily fluids

In vitro studies on the pathogenesis of the human cytomegalovirus (HCMV) are conducted regularly using laboratory adapted strains that lose some characteristics during the adaptation process. Since HCMV is excreted from bodily fluids during infection or reactivation, this work aimed to isolate and culture HCMV from the MRC-5 human cells found in the urine, bronchoalveolar lavage, saliva, and plasma samples of pediatric patients with probable or confirmed infection. The samples were inoculated on cell cultures either for 14 days or until a cytopathic effect (CPE) of 80 % was observed. The cell lysates and supernatants were used to perform successive viral passages. Besides HCMV, the herpes simplex virus was detected from all the saliva samples. Inoculation of the HCMV positive sera induced cell clustering and immediate monolayer damage that restricted their use. One sample of bronchoalveolar lavage induced a CPE after inoculation like that of the HCMV reference strains (Towne and Merlin), which was consequently propagated and titrated. A second viral isolate derived from the urine sample of a patient with congenital infection did not demonstrate a CPE, although presence of the virus had been confirmed using PCR. The viral isolates were examined and found to be negative for adenoviruses or enteroviruses. Despite the evident difficulty encountered for the isolation and harvesting of the HCMV, this work shows that it was possible to obtain a low passage viral strain using a modified shell vial method and inoculation protocol with extended follow-up and confirmation

Sobrerregulación de ciclooxigenasa en respuesta inflamatoria de fibroblastos gingivales infectados con citomegalovirus

Objetivos. Evaluar los cambios de expresión de COX-1 y COX-2 en fibroblastos gingivales infectados con citomegalovirus. Métodos. Se cultivaron fibroblastos gingivales humanos primarios criopreservados y se confirmó su fenotipo mesenquimal mediante inmunofluorescencia para vimentina. Posteriormente se infectaron a una multiplicidad de infección (MOI) de 0,5 con dos cepas de CMV: una cepa de laboratorio (Towne) y un aislado clínico (B52). Las células se recolectaron a las 24 h y 48 h después de la infección (p.i) para la extracción total de ARN. La cuantificación relativa de ARNm para COX-1 y COX-2 se evaluó mediante RTqPCR utilizando sondas de hidrólisis. Simultáneamente, se realizó inmunofluorescencia para COX-2 en células infectadas. Resultados. La infección por CMV de los fibroblastos gingivales produjo a las 24 h p.i. un aumento de 1,7 y 7,3 veces de transcritos para COX-2 en células infectadas con la cepa Towne y el aislado clínico B52 respectivamente, mientras que para el ARNm de COX-1, B52 provocó un aumento de 2,3 veces. A las 48 h p.i, el efecto citopático no permitió recolectar el monocapa celular. La inmunofluorescencia para COX-2 fue positiva en citoplasma y región perinuclear en células infectadas. Conclusiones. La infección tanto con el aislado clínico B52 como con la cepa Towne, indujo sobrerregulación de COX-2, sin embargo, B52 indujo niveles más altos en comparación con Towne. Se evidencio expresión perinuclear y citoplasmática de COX-2 asociada con núcleos atípicos

A Case Series of Severe Dengue with Neurological Presentation in Children from a Colombian Hyperendemic Area

Dengue transmission is sustained in Colombia with increasing prevalence mainly in children. This work aimed to describe a case series of children diagnosed with dengue presenting neurological disease in Huila Province of Colombia. Eleven pediatric febrile patients confirmed for dengue disease and presenting neurological signs were studied in the University Hospital of Neiva, Huila Province. Clinical and laboratory findings, CSF cytochemical analysis, neurology images, and serology and molecular studies were performed. Viral RNA was detected in all patients’ sera by RT-PCR. Nine out of 11 were primary infections. Tonic-clonic seizures (73%), consciousness alterations (27%), irritability (27%), and ataxia (18%) were the most frequent neurological signs. None of the patients had plasma leakage, hypovolemic shock, or liver disease, confirming the encephalitis diagnosis. Diagnostic images did not show abnormal findings, but neither bacterial nor fungal infections were detected in CSF analysis. All patients survived without sequelae except for one patient that presented ataxia for months. In conclusion, we described a group of children with neurological signs during severe dengue disease as the main finding, indicating the importance to including dengue as a differential diagnosis in neurological patients from endemic areas

Prevalence of dengue antibodies in healthy children and adults in different Colombian endemic areas

Objectives Colombia is a dengue hyperendemic country; however, the prevalence of antibodies against dengue in the general population including the inhabitants of rural areas is unknown. This study aimed to determine the prevalence of dengue IgM and IgG antibodies in healthy children and adults in urban and rural areas of seven different endemic regions in Colombia between 2013 and 2015. Design or method Blood samples from healthy volunteers (1,318) were processed by serology (by indirect IgG and capture IgM and IgG ELISA) and molecular tests to detect viral RNA and circulating serotypes. Results The seroprevalence of IgG for dengue were 85% in children and over 90% for adults. In addition to the high IgM positive rate (14.9%) and secondary recent infection marker rate (capture IgG, 16%), 8.4% of the healthy volunteers were positive for dengue virus (DENV) RNA. Conclusion This study confirmed the broad and permanent circulation of DENV in Colombia and the high rates of infection and reinfection suffered by its inhabitants. This information can be used by the health authorities to strengthen vector control and vaccine policies and review the algorithms of diagnosis and disease management in children and adults